Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support

Kinetics of proton transport into influenza virions by the viral M2 channel

M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pKm of ~4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer

\u3ci\u3eToxoplasma gondii\u3c/i\u3e requires its plant-like heme biosynthesis pathway for infection

Heme, an iron-containing organic ring, is essential for virtually all living organisms by serving as a prosthetic group in proteins that function in diverse cellular activities ranging from diatomic gas transport and sensing, to mitochondrial respiration, to detoxification. Cellular heme levels in microbial pathogens can be a composite of endogenous de novo synthesis or exogenous uptake of heme or heme synthesis intermediates. Intracellular pathogenic microbes switch routes for heme supply when heme availability fluctuates in their replicative environment throughout infection. Here, we show that Toxoplasma gondii, an obligate intracellular human pathogen, encodes a functional heme biosynthesis pathway. A chloroplast-derived organelle, termed apicoplast, is involved in heme production. Genetic and chemical manipulation revealed that de novo heme production is essential for T. gondii intracellular growth and pathogenesis. Surprisingly, the herbicide oxadiazon significantly impaired Toxoplasma growth, consistent with phylogenetic analyses that show T. gondii protoporphyrinogen oxidase is more closely related to plants than mammals. This inhibition can be enhanced by 15- to 25-fold with two oxadiazon derivatives, lending therapeutic proof that Toxoplasma heme biosynthesis is a druggable target. As T. gondii has been used to model other apicomplexan parasites, our study underscores the utility of targeting heme biosynthesis in other pathogenic apicomplexans, such as Plasmodium spp., Cystoisospora, Eimeria, Neospora, and Sarcocystis

Redistribution of Nickel Ions Embedded within Indium Phosphide Via Low Energy Dual Ion Implantations

Transition-metal doped Indium Phosphide (InP) has been studied over several decades for utilization in optoelectronics applications. Recently, interesting magnetic properties have been reported for metal clusters formed at different depths surrounded by a high quality InP lattice. In this work, we have reported accumulation of Ni atoms at various depths in InP via implantation of Ni- followed by H– and subsequently thermal annealing. Prior to the ion implantations, the ion implant depth profile was simulated using an ion-solid interaction code SDTrimSP, incorporating dynamic changes in the target matrix during ion implantation. Initially, 50 keV Ni- ions are implanted with a fluence of 2 × 1015 atoms cm-2, with a simulated peak deposition profile approximately 42 nm from the surface. 50 keV H- ions are then implanted with a fluence of 1.5 × 1016 atoms cm-2. The simulation result indicates that the H- creates damages with a peak defect center ~400 nm below the sample surface. The sample has been annealed at 50°C in an Ar rich environment for approximately 1hr. During the annealing, H vacates the lattice, and the formed nano-cavities act as trapping sites and a gettering effect for Ni diffusion into the substrate. The distribution of Ni atoms in InP samples are estimated by utilizing Rutherford Backscattering Spectrometry and X-ray Photoelectron Spectroscopy based depth profiling while sputtering the sample with Ar-ion beams. In the sample annealed after H implantation, the Ni was found to migrate to deeper depths of 125 nm than the initial end of range of 70 nm

Bostonia: The Boston University Alumni Magazine. Volume 20

Founded in 1900, Bostonia magazine is Boston University's main alumni publication, which covers alumni and student life, as well as university activities, events, and programs

c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae.

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization

Adaptive capacity beyond the household: a systematic review of empirical social-ecological research

The concept of adaptive capacity has received significant attention within social-ecological and environmental change research. Within both the resilience and vulnerability literatures specifically, adaptive capacity has emerged as a fundamental concept for assessing the ability of social-ecological systems to adapt to environmental change. Although methods and indicators used to evaluate adaptive capacity are broad, the focus of existing scholarship has predominately been at the individual- and household- levels. However, the capacities necessary for humans to adapt to global environmental change are often a function of individual and societal characteristics, as well as cumulative and emergent capacities across communities and jurisdictions. In this paper, we apply a systematic literature review and co-citation analysis to investigate empirical research on adaptive capacity that focus on societal levels beyond the household. Our review demonstrates that assessments of adaptive capacity at higher societal levels are increasing in frequency, yet vary widely in approach, framing, and results; analyses focus on adaptive capacity at many different levels (e.g. community, municipality, global region), geographic locations, and cover multiple types of disturbances and their impacts across sectors. We also found that there are considerable challenges with regard to the ‘fit’ between data collected and analytical methods used in adequately capturing the cross-scale and cross-level determinants of adaptive capacity. Current approaches to assessing adaptive capacity at societal levels beyond the household tend to simply aggregate individual- or household-level data, which we argue oversimplifies and ignores the inherent interactions within and across societal levels of decision-making that shape the capacity of humans to adapt to environmental change across multiple scales. In order for future adaptive capacity research to be more practice-oriented and effectively guide policy, there is a need to develop indicators and assessments that are matched with the levels of potential policy applications

Kinetics of Proton Transport into Influenza Virions by the Viral M2 Channel

M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pKm of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer

Automated Computational Detection of Disease Activity in ANCA-Associated Glomerulonephritis Using Raman Spectroscopy: A Pilot Study

Biospectroscopy offers the ability to simultaneously identify key biochemical changes in tissue associated with a given pathological state to facilitate biomarker extraction and automated detection of key lesions. Herein, we evaluated the application of machine learning in conjunction with Raman spectroscopy as an innovative low-cost technique for the automated computational detection of disease activity in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis (AAGN). Consecutive patients with active AAGN and those in disease remission were recruited from a single UK centre. In those with active disease, renal biopsy samples were collected together with a paired urine sample. Urine samples were collected immediately prior to biopsy. Amongst those in remission at the time of recruitment, archived renal tissue samples representative of biopsies taken during an active disease period were obtained. In total, twenty-eight tissue samples were included in the analysis. Following supervised classification according to recorded histological data, spectral data from unstained tissue samples were able to discriminate disease activity with a high degree of accuracy on blind predictive modelling: F-score 95% for >25% interstitial fibrosis and tubular atrophy (sensitivity 100%, specificity 90%, area under ROC 0.98), 100% for necrotising glomerular lesions (sensitivity 100%, specificity 100%, area under ROC 1) and 100% for interstitial infiltrate (sensitivity 100%, specificity 100%, area under ROC 0.97). Corresponding spectrochemical changes in paired urine samples were limited. Future larger study is required, inclusive of assigned variables according to novel non-invasive biomarkers as well as the application of forward feature extraction algorithms to predict clinical outcomes based on spectral features

Genomewide Association Study of Statin-Induced Myopathy in Patients Recruited Using the UK Clinical Practice Research Datalink.

Statins can be associated with myopathy. We have undertaken a genomewide association study (GWAS) to discover and validate genetic risk factors for statin-induced myopathy in a "real-world" setting. One hundred thirty-five patients with statin myopathy recruited via the UK Clinical Practice Research Datalink were genotyped using the Illumina OmniExpress Exome version 1.0 Bead Chip and compared with the Wellcome Trust Case-Control Consortium (n = 2,501). Nominally statistically significant single nucleotide polymorphism (SNP) signals in the GWAS (P T in the SLCO1B1 gene) SNP was genomewide significant in the severe myopathy (creatine kinase > 10 × upper limit of normal or rhabdomyolysis) group (P = 2.55 × 10-9 ; odds ratio 5.15; 95% confidence interval 3.13-8.45). The association with SLCO1B1 was present for several statins and replicated in the independent validation cohorts. The data highlight the role of SLCO1B1 c.521C>T SNP as a replicable genetic risk factor for statin myopathy. No other novel genetic risk factors with a similar effect size were identified